Translocation, phosphorylation and association of signaling proteins in membrane microdomains, strongly suggest individual imperative roles of PKC1 and RhoA in colonic motility. Phosphorylation of HSP27 is essential for translocation of PKC1 and RhoA during contraction of colonic circular smooth muscle cells (CSMC). Contraction of CSMC is associated with HSP27 phosphorylation, while HSP20 phosphorylation at S16 inhibits contraction. Preliminary results from adult CSMC show: 1) the presence of cav-1 in lipid raft membrane fractions;2) increased acetylcholine (Ach)-induced sequestration of PKC1, phospho-PKC1 (S657) and HSP27 into the lipid rafts and translocation of HSP20 out of the lipid rafts;3) increased association of PKC1 and HSP27 with caveolin 1 (cav-1) in the particulate fraction of CSMC;and 4) silencing of PKC1 (siRNA for PKC1) had an inhibitory effect of both the initial rise and the sustained phase of Ach-induced force generation in 3-dimensional rings bioengineered (3DBR) from human colon CSMC treated with siRNA for PKC1 suggesting a role for PKC1 in force generation and its maintenance. Preliminary data from aged rat CSMC show that isolated lipid raft fractions were depleted of cav-1 and consequently of phospho-PKC1 (S657) concomitant with decreased association of PKC1 and HSP27 with cav-1. Further, adult CSMC transfected with DN cav-1 cDNA, mimicked Ach response of aged CSMC by exhibiting reduced association of PKC1 and HSP27 with cav-1. This correlated with reduced Ach-induced force generation of 3DBR from CSMC of aged rats. These data suggest a crucial role for lipid rafts, cav-1, and HSP27 in normal contractile responses and a reduction in caveolae formation associated with aging CSMC. Reduced caveolae formation could be a critical factor affected by aging and a putative therapeutic target. Preliminary data indicates that ectopic expression of wt-cav-1 reinstated Ach-induced force generation in 3DBR from aged colon. Therefore, we propose to use multilevel functional approaches to study the intricacies of contractile signaling pathways. We will study the spatiotemporal reorganization and relocation of different proteins using live cell imaging, biochemical and molecular biology tools and real time physiological monitoring of contractile response using 3-dimensional rings bioengineered from adult, aged, and stably transfected smooth muscle cells. The data obtained through these approaches will allow us to: 1) Discern the intricate molecular mechanisms responsible for the understanding of the physiology of colonic motility;2) Understand and identify putative disrupted mechanisms affected by aging that contribute to the sluggishness and pathophysiology of contraction of the colon;and 3) Identify and test the possible putative targets to rectify age-related pathophysiology and sluggishness of colonic motility. Public Health Relevance: In summary, we will utilize biochemical, molecular and structural physiological tools we have developed to detect the disruption of normal physiological motor function due to aging and ultimately design therapies to rectify these defects.